It has been recognized that recent progress in genetic engineering offers plant breeders the ability to avoid the delay in crop improvement inherent in classical breeding techniques. However, there remains a difficulty in application of these techniques, for unicellular and multicellular organisms require different techniques to change the entire genetic makeup. In microorganisms, one attempts to effect change at the cellular level with confidence that this will be reproduced through succeeding generations.
In organisms which are not normally unicellular, such as plants, it is advantageous to perform genetic manipulations at the cellular level, then regenerate and raise a mature plant expressing the new characteristics. The cloning of plants by asexual means offers advantages to plant breeders which cannot be obtained using sexual means of reproduction. Sexual reproduction necessitates the recombination of traits or characters in such a way that often, the sib generation loses the properties of its parents. Cloning of plants allows the breeder to reproduce and expand the numbers of individuals all of which have the same genetic make-up.
Plants can be cloned by a variety of methods other than somatic embryogenesis such as by cuttings, air layering, tuber and close division and in vitro micropropagation. All of these methods are labor intensive and may be economically disadvantageous for some applications. Cloning by somatic embryogenesis can offer high numbers of clones (20,000 to 40,000 units per gram fresh weight input) and low cost since embryos can be produced in batch suspension culture and handled automatically in a fluid medium.
In vitro cultivation of plant tissue requires that the tissue be maintained in a medium which provides nutrition and sustains viability. This tissue maintenance can be promoted in plant organ, tissue or cell cultures.
In culture, plant cells are typically induced to undergo repeated cell divisions on a nutritive substrate, producing an amorphous cell mass known as callus. The callus can be maintained through subculture to allow mass proliferation. The callus may also be induced to undergo differentiation which results in the organized tissues and organs of the mature plant.
In this manner, genetic changes may be effected on a cellular level and then maintained through subsequent development to produce an entire crop with identical genetic characteristics. This allows the plant breeder to bypass the normal genetic barriers in plant reproduction, and obtain a more uniform and advantageous field crop.
Somatic embryogeneis can be accomplished in numerous plant species. Examples of plants capable of somatic embryogenesis are cited in Evans, D. A. et al., "Growth and Behavior of Cell Cultures: Embryogenesis and Organogenesis" in Plant Tissue Culture: Methods and Applications in Agriculture, T. A. Thorpe, Ed., Academic Press, pg. 45 et seq. (1981). These examples are not exhaustive; reports of new species capable of somatic embryogenesis appear weekly.
Technology reviews on somatic embryogenesis have outlined methods which enable production of somatic embryos (see Kohlenback, H. W., "Comparative Somatic Embryogenesis," in Frontiers of Plant Tissue Culture, T. A. Thorpe, pg, 59 et. seq., Int. Plant Tissue Cult. Congress, Calgary, CN. (1978); Sharp et al. "The Physiology of in vitro Asexual Embryogenesis" Hortic. Reviews 2, 268-310 (1982)). One parameter which is necessary for embryogenesis from somatic cell cultures is a pretreatment with an auxin. One auxin which is generally used in 2,4-dichlorophenoxyacetic acid (2,4-D) although this is by no means to only auxin which can be utilized. A detailed investigation of auxin activity for carrot somatic embryogenesis has been done by Kamada, H. and H. Harada "Studies on the Organogenesis in Carrot Tissue Culture. I. Effects of Growth Regulators on Somatic Embryogenesis and Root Formation." Zeit. Pflanzenphysiol. 91, 255. 1979, and in alfalfa by Walker, K. A. et al., "Initiation of Sexual Embryogenesis in Somatic Cell Cultures," Tissue Culture Assn. Meeting Abstracts, No. 68, (1980) and Walker, K. A. et al., " Organogenesis in Callus Tissue of Medicago sativa. The Temporal Separation of Induction Processes from Differentiation Processes," Plant Sci. Lett. 16, 23-30 (1979).
Some techniques exist for increasing the quantity of the embryonic cells obtained through tissue culture of plant somatic tissue. A requirement for a source of reduced nitrogen for the formation of in vitro somatic embryos has been recognized in carrot cells cultures. This effect has since been confirmed and extended to a variety of other species. A detailed study of this reduced nitrogen requirement may be found in Walker, K. A. and S. J. Sato "Morphogenesis in Callus Tissue of Medicago sativa: "The Role of Ammonium Ion in Somatic Embryo Genesis," Plant Cell Tissue Organ Culture, 1: 109-21.
It has also been recognized that certain amino acids will stimulate somatic embryogenesis in carrot cell cultures. Wetherell, D. F. and D. K. Dougall "Sources of Nitrogen Supporting Growth in Embryogenesis in Cultured Wild Carrot Tissue," Physiol. Plant. 37: 97-103 and Kamada, H. and H. Harada, "Studies on the Organogenesis in Carrot Tissue Cultures II, Effects of Amino Acids and Inorganic Nitrogenous Compounds on Somatic Embryogenesis", Z. Pflanzenphysiol. 91: 453-463. (1979). However, previous studies have either lacked sufficient control treatments to allow comparison to other experimental treatments or they are limited to a small range of concentrations which so not test for optimization.
The auxin studies previously cited have defined three parameters which interact in the induction of embryogenesis by auxin. These are: (1) auxin structure; (2) auxin concentration; and (3) time of exposure. For example, Kamada and Harada find that auxin structure determines potency in affecting differentiation in cell culture. The lower the concentration threshold for auxin-induced differentation, the higher the potency. The reported order of decreasing potency for auxin for somatic embryogenesis in carrot was 2,4,5-trichlorophenoxyacetic acid=m-chlorophenoxyacetic acid&gt;2,4-dichlorophenoxyacetic acid&gt;p-chlorophenoxyacetic acid&gt;.alpha.-naphthylene acetic acid=indole-3-acetic acid&gt;indole-3-butyric acid. Differences in potency due to auxin structure can be compensated for by adjusting auxin concentration to achieve comparable regeneration. Likewise, time of exposure to a given auxin structure at a given concentration is a critical for efficient regeneration. The product of auxin structure, auxin concentration and time of exposure will be referred to hereafter as dosage (dose) of auxin or auxin dosage (dose).
A dosage of auxin usually precedes somatic embryogenesis from cell culture. Embryo differentiation occurs when cells grown at one auxin dosage (primary dosage) are either subcultured on medium containing a lower auxin dosage or no auxin, or treated by other methods which decrease auxin dosage (see Kohlenbach review). Continued exposure of cells to the primary auxin dosage is not recommended by numerous authors (Phillips and Collins, Crop Sci., 20, 323, 1980; Kohlenbach (1978) as before; Sharp et al. (1980) as before, Reinert, J. and M. Tazawa, Planta 87, 239, 1969 and Halperin, W. Science 146, 408, 1964) as embryo regeneration is disrupted by these dosages.
Another factor which enables embryogenesis from somatic cell cultures is the addition of reduced nitrogen to the culture medium. This nitrogen requirement can be fulfilled by ammonium, a variety of L-amino acids, or combinations of all of these (Halperin, W. and D. F. Wetherell, Nature 205, 519-1965; Wetherell, D. F. and D. K. Dougall, Physiol. Plant 37, 97-1976; Kamada, H. and H. Harada. Z. Pflanzenphysiol. 91, 453 (1979). This requirement for reduced nitrogen can be supplied prior to auxin removal or decrease in auxin dosage (Halperin, W. Amer. J. Bot. 53, 443. 1966) or in the regeneration medium containing no or reduced auxin dosage. This yields high frequency and high quality embryos of asexual origin.
Several authors have recognized that auxin and ammonium ion interact in somatic embryogenesis (Sharp et al. (1980), Halperin, Am. J. Bot. 53: 443-453 1966, Caldas, L. S. Effects of various growth hormones on the production of embryoids from tissue culture of the wild carrot, Daucus carota L., Ph.D. dissertation, Ohio State Univ., 1971). These authors recommend against the use of 2,4-D or other auxins as an additive to plant regeneration medium. Sharp et al. (1980) point out that ammonium ion may merely permit somatic embryogenesis to occur after pretreatment with higher auxin dosages.
It is an object of this invention to provide methods and materials to increase the quantity of somatic embryos produced from plant tissue.
It is a further object of this invention to provide optimized sources of reduced nitrogen in combination with auxins for regeneration of somatic embryos.
It is yet another object of this invention to provide methods and materials allowing mass propagation of numerous species of plants through somatic embryogenesis.
It is a still further object of this invention to provide methods and materials for a generation of numerous viable somatic embryos with identical genetic and phenotypic traits.